We propose to further understand the biochemical basis of the requirement of many malignant and transformed cells for exogenous methionine (Met) despite high levels of endogenous Met synthesis from homocysteine (Hc). We shall in Met dependent (Met minus) and independent cells (Met plus) kinetically study the formation of the cellular free Met pool from Met precursors as well as the utilization of Met from this pool to form the two major metabolites of Met, S-adenosylmethionine (SAM) and Met-tRNA. We will do these studies comparatively in Met plus normal human diploid cells, Met minus transformed and malignant cells, and Met plus cells derived from them. We have found that, when Met minus cells are incubated in Hc medium, they are reversibly growth arrested, possibly at thA G1/S border of the cell cycle, which we will further characterize with respect to position in cell cycle and which factors are rate limiting. We have seen in human cells, including normal human diploid fibroblasts, that the biosynthesis of the two enzymes catalyzing the major reactions utilizing Met, methionine adenosyltransferase (MAT) and methionyl ion RNA synthetase (MTS), are repressed by Met. In normal human diploid cells MAT derepresses only in growing cells, not resting cells. In Met minus cells endogenously synthesized Met does not seem to repress MAT. We propose to further understand the genetic regulation of these enzymes, their relation to growth control, and normal and altered cellular compartmentation of Met. We have used the Met dependence marker in transformed and malignant cells to select Met plus cells which have concomitantly reverted for properties of transformation including the first human transformation revertants. We shall utilize these diverse revertants to ascertain in collaborative studies with D. Livingston whether their properties are correlated with various forms of SV40 specific T-antigen.